We compared trimethoprim sulfate 0.1%/polymyxin B sulfate 10,000 units/mL with tobramycin 0.3% for preoperative sterilization of the ocular surface, aqueous humor concentration, and ocular safety and comfort in 99 patients who had cataract extraction and intraocular lens implantation. The organisms most frequently cultured from the conjunctiva at baseline were Staphylococcus epidermidis, Corynebacterium species, and Staphylococcus aureus, which were isolated from 66%, 15%, and 8% of the 95 specimens eligible for evaluation. All organisms identified in positive baseline conjunctival cultures except Staphylococcus epidermidis were completely eradicated in both groups on the day of surgery and five to seven days postoperatively. Staphylococcus epidermidis was eradicated on the day of surgery in 58% of patients in the trimethoprim/polymyxin group and in 68% in the tobramycin group. This organism was eradicated five to seven days postoperatively in 85% of patients in both groups. Mean aqueous humor concentration of trimethoprim sulfate at surgery was greater than the mean tobramycin concentration, but neither reached clinically significant inhibitory levels for most organisms. No significant differences were found in ocular safety and comfort.